The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing. Alterations in this essential step in eukaryotic gene expression are known to underly many human diseases, so detailed understanding of the mechanisms involved is important for the betterment of human health. Pre- mRNA splicing is carried out by the spliceosome, a ~3 MDa macromolecular complex consisting of 5 small nuclear RNAs (snRNAs: U1, U2, U4, U5 and U6) and >100 polypeptides. While much progress has been made in defining these component parts, much remains to be learned about how these myriad pieces function together to mediate precise and timely intron removal. This proposal will apply two new enabling methodologies, RIPiT-Seq (RNA IP in Tandem combined with Deep Sequencing) and CoSMoS (Colocalization Single Molecule Spectroscopy), to study mammalian spliceosome assembly in vivo and in vitro. RIPiT-Seq allows researchers to map in vivo occupancy sites of multicomponent RNPs of defined composition across the entire transcriptome. By varying the pairs of proteins used for IP and/or affinity purification +/- formaldehyde crosslinking one can determine the complete occupancy landscapes for different complexes, as well as which binding sites are kinetically stable and which are more dynamic. At the other end of the spectrum, CoSMoS enables real time observation of spliceosome assembly dynamics on single pre-mRNA molecule. The proposed experiments will address numerous key questions including: What is the spliceosome assembly state on retained and slowly processed introns? Are alternative splicing decisions strictly limited to early stage complexes, or do some decisions involve later stage complexes? Are there any sites of mammalian spliceosome catalysis at sites other than currently defined intron ends? Is mammalian spliceosome assembly as dynamic as yeast spliceosome assembly? In what way are spliceosome assembly pathways altered to favor inclusion or exclusion of cassette exons upon large-scale transcriptional reprogramming? Together these experiments will fundamentally change our understanding of the basic pathways of mammalian spliceosome assembly and how these pathways are altered to tune gene expression output in response to external stimuli.